The membranes found at the surface of mammalian cells perform functions of great importance relating to the integrity and activities of cells and tissues. Of particular interest is the study of ion channel biochemistry, physiology, pharmacology and biokinetics. These ion channels, which include sodium (Na.sup.+), potassium (K.sup.+) and calcium (Ca.sup.2+) channels are present in all mammalian cells and control a variety of physiological and pharmacological processes.
Potassium channels are implicated in a broad spectrum of processes in excitable and non-excitable cells. These physiologic processes include regulation of heartbeat (Breitwieser, G. E. and Szabo, G. (1985) Nature 317:538, Logothetis, D. E. et al. (1987) Nature 325:321, Yatani, A. et al. (1987) Science 235:207, Yanti, A. et al. (1988) Nature 336:680, Brown, A. M. and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52:197 and Kurachi, Y. et al. (1992) Progress in Neurobiol. 39:229), dilation of arteries (Nelson, M. T. et al. (1990) Nature 344:770), release of insulin (Rorsman, P. et al. (1991) Nature 349:77), excitability of nerve cells (Stanfield, P. R. et al. (1985) Nature 315:498, Williams, J. T. et al. (1988) J. Neurosci. 8:3499) and regulation of renal electrolyte transport (Wang, W. et al. (1992) Annu. Rev. Physiol. 54:81).
Several classes of K.sup.+ channels have been identified based on their pharmacological and electrophysiological properties; these include voltage-gated, ATP-sensitive, muscarinic-activated, S type, SK Ca.sup.2+ -activated, Na.sup.+ -activated and inward rectifier types of K.sup.+ channels (For review see Hille, B. Ionic Channels of Excitable Membranes 2d ed., Sinauer, Sunderland, Mass., 1992).
The best characterized class of K.sup.+ channels are the voltage-gated K.sup.+ channels. The prototypical member of this class is the protein encoded by the Shaker gene in Drosophila melanogaster (Papazian, D. M. et al. (1987) Science 237:749, Tempel, B. L. et al. (1987) Science 237:770 and Baumann, A. et al. (1987) EMBO J. 6:3419). Mammalian homologues of the Drosophila Shaker and related Shal, Shab and Shaw genes have been cloned (Wei, A. et al. (1990) Science 248:599, Tempel, B. L., Jan, Y. N. and Jan, L. Y. (1988) Nature 332:837, Baumann, A. et al. (1988) EMBO J. 7:2457, Frech, G. C. et al. (1989) Nature 340:642, Yokoyama, S. et al. (1989) FEBS Lett. 259:37, Cristie, M. J. et al. (1989) Science 244:221, Stuhmer, W. et al. (1989) EMBO J. 8:3235, Swanson, R. et al. (1990) Neuron 4:929 and Luneau, C. J. et al. (1991) Proc. Natl. Acad. Sci. USA 88:3932). Voltage-gated K.sup.+ channels belong to the superfamily of voltage-gated and second messenger-gated cation channels (Jan L. Y. and Jan, Y. N. (1992) Cell 69:715 and Jan, L. Y. and Jan, Y. N. (1990) Nature 345:672). The proteins in this gene family contain one or four copies of an underlying structural motif characterized by six membrane-spanning segments (S1-S6), a putative voltage sensor (S4) and an S5-S6 linker (H5 or P region) involved in ion conduction. The vast majority of cloned K.sup.+ channels share a structural organization to the above motif, thereby placing most of the cloned K.sup.+ channels in the same K.sup.+ channel superfamily. Only two cloned K.sup.+ channels do not share the above structural organization. These are the mink channel (Takumi, T. et al. (1988) Science 242:1042) and the ROMK1 channel, an ATP-regulated K.sup.+ channel (Ho, K. et al. (1993) Nature 362:31). Attempts to isolate inward rectifier K.sup.+ channels using sequences derived from members of the voltage-gated K.sup.+ channel gene family as probes have been unsuccessful. This suggests that the structural organization of the inward rectifier channels differs significantly from that of the voltage-gated K.sup.+ channels.
The molecular features of the proteins which comprise the classes of K.sup.+ channels which are not voltage-gated are, for the most part, unknown although pharmacological and physiological characteristics have been elucidated. Of particular interest are the inward rectifier K.sup.+ channels.
Inward rectifier K.sup.+ channels allow primarily K.sup.+ influx but little K.sup.+ outflux. These K.sup.+ channels have been found in a variety of cell types including skeletal (Katz, B. (1949) Arch. Sci. Physiol. 2:285 and Standen, N. B. and Stanfield, P. R. (1978) J. Physiol. 280:169) and cardiac (Sakmann, B. and Trube, G. (1984) J. Physiol. 347:641) muscle cells, starfish and tunicate oocytes (Hagiwara, S. et al. (1976) J. Gen. Physiol. 67:621 and Okamoto, H. et al. (1976) J. Physiol. 254:607), neurons (Mayer, M. L. & Westbrook, G. L. (1983) J. Physiol. 340:19, Mihara, S. et al. (1987) J. Physiol. 390:335, Inoue, M. et al. (1988) J. Physiol. 407:177 and Williams, J. T. et al. (1988) J. Neurosci. 8:3499), glial cells (Barres, B. A. (1991) Current Opinion in Neurobiol. 1:354), blood cells (McKinney, L. C. and Gallin, E. K. (1988) J. Memb. Biol. 103:41 and Lewis, D. L. et al. (1991) FEBS Lett. 290:17) and endothelial cells (Silver M. R. & DeCoursey T. E. (1990) J. Gen. Physiol. 96:109).
The inward rectifier K.sup.+ channels have significant roles in maintaining the resting potential and in controlling excitability of a cell. The physiological functions of the inward rectifier K.sup.+ channels stem from their unique rectification property and consist of three parts (Hille, B. Ionic Channels of Excitable membranes, 2d ed., Sinauer, Sunderland, Mass., 1992). First, the absence of outward conductance at highly depolarized membrane potentials allows a cell that expresses predominantly the inward rectifier to maintain prolonged depolarization. This is important for the generation of prolonged action potentials in heart ventricular cells, and for the prevention of double fertilization of oocytes (Hagiwara, S. and Jaffe, L. A. (1979) Annu. Rev. Biophys. Bioeng. 8:385). Second, the large inward conductance at membrane potentials below the K.sup.+ equilibrium potential (E.sub.K) prevents excessive hyperpolarization, which may be caused by the electrogenic Na.sup.+ pump (Hille, B., supra). Third, the slight outward conductance of inward rectifier K.sup.+ channels at membrane potentials just above E.sub.K helps to keep the resting membrane potential close to E.sub.K. Modulation of this conductance level changes the resting potential and alters the excitability of the cell. For example, it is well known that the activation of a particular type of inward rectifier K.sup.+ channel, the muscarinic K.sup.+ channel, by acetylcholine causes hyperpolarization of the cardiac pacemaker cells and slows the heartbeat (Noma, A. et al. (1979) Pflugers Arch. 381:255).
The inward rectification properties essential for the physiological functions of these K.sup.+ channels have been characterized at the mechanistic level. These channels are permeable to an inward flow of K.sup.+ ions at membrane potentials below E.sub.K, which may be varied by changing the extracellular K.sup.+ ion concentration (Hagiwara, S. et al. (1976) J. Gen. Physiol. 67:621). Therefore, the inward rectifier K.sup.+ channels do not activate over a fixed range of membrane potentials, unlike voltage-gated K.sup.+ channels. The inward rectification has been shown to be mainly due to the blockade of outward current by internal Mg.sup.2+ ; in the absence of Mg.sup.2+, inward rectifier K.sup.+ channels exhibit a linear current-voltage relation (Matsuda, K. et al. (1987) Nature 325:156, Vandenberg, C. A. (1987) Proc. Natl. Acad. Sci. USA 84:2560 and Matsuda, H. (1988) J. Physiol. 397:237).
The extensive interaction of the inward rectifier K.sup.+ channel pore with permeant ions and blocking ions has been well documented. These studies reveal that the inward rectifier K.sup.+ channel has a long-pore with multiple binding sites; permeant ions enter the pore in single file and exhibit discernible interactions with other ions that either permeate or block the pore (Hille, B., supra, Hagiwara, S. et al. 1977) J. Gen. Physiol. 70:269 and Ohmori, H. (1980) J. Memb. Biol. 53:143). Interactions between permeant ions are manifested by the fact that K.sup.+ conductance of the inward rectifier does not increase linearly with K.sup.+ concentration (Sakmann, B. and Trube, G., supra). Extracellular cations such as barium (Ba.sup.2+) and cesium (Cs.sup.+) block the inward rectifier in a manner that depends both on the voltage and on the time elapsed following channel activation. This suggests that these blocking ions enter the open channel pore so that they sense part of the voltage drop across the membrane (Hagiwara, S. et al. (1976) J. Gen. Physiol. 67:621). The steepness of this voltage dependence of the block further indicates that there are multiple binding sites for Cs.sup.+ in the channel pore (Hagiwara, S. et al. (1976) J. Gen. Physiol. 67:621 and Hille, B., supra).
Ion channel function can be regulated by various substances, such as hormones and neurotransmitters, via specific membrane receptors. Two major categories of receptor-operated ion channels are known. One class consists of ion channels which have an intrinsic sensor; the receptor site and a channel pore are present in the same polypeptide. The other class consists of ion channels having a remote sensor; the receptor site and the ion channel are different membrane proteins. G Protein is often involved in the remote-sensing ion channel models of transmembrane signalling.
An important receptor involved in the regulation of heart rate, the muscarinic receptor, slows heart rate upon activation by parasympathetic nerve stimulation (Trautwein, W. and Dudel, J. (1958) Pflugers Arch. 266:324, Noma, A. et al. (1979) Pflugers Arch. 381:255, Sakmann, B. et al. (1983) Nature 303:250 and Soejima, M. and Noma, A. (1984) Pflugers Arch. 400:424). The heart rate is slowed by the opening of the muscarinic K.sup.+ channel. This ion channel has a remote sensor as the muscarinic receptor and K.sup.+ channel exist as separate protein molecules.
The muscarinic K.sup.+ channels in the sinoatrial node and atrium, the pacemaker of the heart, are inward rectifying K.sup.+ channels and are known to be directly coupled with G proteins (Breitwieser, G. E. and Szabo, G. (1985) Nature 317:538, Logothetis, D. E. et al. (1987) Nature 325:321, Latani, A. et al. (1987) Science 235:207, Latani, A. et al. (1988) Nature 336:680, Brown, A. M. and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52:197 and Kurachi, Y. et al. (1992) Progress in Neurobiol. 39:229). G proteins are a class of proteins involved in intracellular signal transduction. The G protein senses when a ligand has occupied a cell surface receptor, binds GTP and activates another protein involved in the signal transduction pathway such as adenyl cyclase or an ion channel. In the case of the muscarinic receptor, the activated G protein opens the muscarinic K.sup.+ channel causing an outflux of K.sup.+ from the heart muscle cell.
While mechanistic studies have elucidated physiological and pharmacological properties of the muscarinic K.sup.+ channel, no studies have been possible at a molecular level. The molecular cloning of the muscarinic K.sup.+ channel would allow the development of assay systems to identify compounds which selectively inhibit this ion channel thereby providing compounds useful for the regulation of heart rate in mammals.
The art needs molecular characterization of inward rectifier K.sup.+ channels in order to elucidate the physiological functions and biophysical properties of these channels. An understanding of these properties will allow the regulation of the physiological functions performed by these K.sup.+ channels, such as regulation of heartbeat and release of insulin. Additionally, the availability of gene sequences encoding these inward rectifier K.sup.+ channels would enable assay systems which would allow the identification of materials capable of selectively blocking these channels. Presently compounds which effect K.sup.+ channels are identified using a cell or a tissue in which multiple types of K.sup.+ channels are present; accordingly it is not possible to determine that a given compound exerts its effect solely through its interaction with a given type of K.sup.+ channel in the present assay. Indeed the K.sup.+ channel modulating drugs currently used to treat physiological disorders mediated by a given class of K.sup.+ channels often have undesirable side effects. The art needs a means to identify compounds which have a specific and selective effect on a single type of K.sup.+ channel for the improved treatment of disease.